Biology subjects

Aizawa, Y.

Publications and source records attributed to Aizawa, Y..

8 recordsLinked to original sources

bioRxiv2026

In-Cell Protein Crystallization via a Locally Flexible 24-mer Assembly Precursor

In-cell protein crystallization (ICPC) produces ordered protein crystals within living cells, but the mechanisms used by proteins to acquire long-range crystalline order in the cellular environment remains poorly understood. Here, we define the assembly pathway of CipB, a crystalline inclusion protein from Photorhabdus luminescens. CipB crystals formed in cells dissolve under mild acidic conditions into a predominant 24-mer species, supporting a model in which an in-cell crystal is built from a discrete 24-mer assembly precursor rather than through direct packing of smaller oligomeric states. Structural analysis of recrystallized CipB shows that the same 24-mer architecture packs into a body-centered cubic lattice, consistent with the lattice observed for the in-cell crystals. Cryo-EM and molecular dynamics analyses indicate that the 24-mer assembly precursor preserves its overall architecture while retaining local conformational flexibility at the N-terminal and surface-loop regions. Mutation analyses further link the N-terminal region to the formation of the 24-mer precursor and surface residues to lattice assembly. These observations support a stepwise crystallization model in which N-terminal flexibility facilitates the formation of an assembly-competent 24-mer precursor, whereas defined hydrophobic surface contacts subsequently organize these precursors into a long-range-ordered lattice.

biophysics↗

bioRxiv2026

Sequence-intrinsic barriers define a class of translation-restricted uORFs in the human genome

Ribosome profiling has revealed widespread translation of upstream open reading frames (uORFs) within the human 5' untranslated region, greatly expanding the apparent coding potential of the genome. However, despite pervasive translational activity, uORF-encoded proteins (uORFps) are only rarely detected by proteomic approaches, creating a major unresolved discrepancy between translation and protein accumulation. Here, we systematically quantified the intracellular accumulation of 111 evolutionarily conserved human uORFps using a unified reporter platform. Although we found that most conserved uORFps accumulated at extremely low levels despite robust transcript expression, protein length emerged as the strongest baseline predictor of this low accumulation. Intriguingly, a subset of conserved uORFs remained poorly accumulated even after experimental extension with a C-terminal EGFP tag, defining a distinct class of translation-restricted uORFs. Sequence and structural analysis of these translation-restricted uORFs revealed a cooperative enrichment of basic amino acids and stable local RNA secondary structures. Synonymous substitutions designed to disrupt these RNA structures substantially restored protein accumulation, demonstrating a major contribution of local transcript stratification to this translation-restricted phenotype. Using these identified rules, we developed a multivariable predictive model that classified 1,432 translation-restricted uORFs across the human transcriptome. Genes harboring these predicted translation-restricted uORFs exhibited significantly reduced downstream translation efficiency and elevated sequence conservation, demonstrating that these sequence-encoded barriers are under purifying selection to act as cis-regulatory elements scattered across the human transcriptome. Together, our findings identify a conserved class of human uORFs that encode intrinsic barriers to productive translation and provide a rigorous framework for understanding how noncanonical coding sequences shape the human proteome.

genetics↗

bioRxiv2025

Functional interplay between condensin I and topoisomerase IIα in single-molecule DNA compaction

Condensin I and topoisomerase II (topo II) are chromosomal ATPases essential for mitotic chromosome assembly. Mechanistically how the two ATPases cooperate to assemble mitotic chromosomes remains unknown. Here we use total internal reflection fluorescence microscopy to analyze the interplay between condensin I and topo II at single-molecule resolution. As observed in previous studies, condensin I alone predominantly forms DNA loops in an ATP-dependent manner. However, when topo II is included in the reaction, condensin I forms stable compact structures (termed "lumps") in a manner dependent on the C-terminal domain of topo II. Each of the stable lumps contains a single condensin I complex and a single topo II dimer. Remarkably, we find that topo II, when catalytically active, renders the lumps resistant to protease treatment. Several lines of evidence show that the protease-resistant lumps contain knotted DNA. A mutant condensin I complex defective in ATP hydrolysis, together with topo II, forms smaller lumps in which the probability of DNA knotting is greatly reduced. Our results demonstrate how topo II-mediated strand passage is coupled with condensin I-mediated loop extrusion to generate a compact DNA structure. Together with recent studies, we discuss the functional implications of these observations in mitotic chromosome assembly and stabilization.

molecular biology↗

bioRxiv2024

Serine chirality guides metabolic flow between one-carbon metabolism and neuromodulator synthesis

O_SCPLOWLC_SCPLOW-serine serves as a central metabolic node that integrates glycolytic flux, lipid metabolism, and one-carbon metabolism. In the mature central nervous system, O_SCPLOWLC_SCPLOW-serine is actively stereo-converted to O_SCPLOWDC_SCPLOW-serine, which functions as a neurotransmitter. However, the role of O_SCPLOWDC_SCPLOW-serine in cellular metabolism remains unclear. Here, we show that O_SCPLOWDC_SCPLOW-serine competes with mitochondrial O_SCPLOWLC_SCPLOW-serine transport, thereby suppressing one-carbon metabolism. Metabolomic analysis revealed that O_SCPLOWDC_SCPLOW-serine reduces intracellular glycine and formate levels, indicating inhibition of the initial step of the one-carbon pathway. Molecular dynamics simulations and enzymatic assays revealed that O_SCPLOWDC_SCPLOW-serine has low affinity for serine hydroxymethyltransferase 2 (Shmt2), which catalyzes the first step in mitochondrial one-carbon metabolism, and does not directly inhibit its activity. Instead, membrane transport assays demonstrated that O_SCPLOWDC_SCPLOW-serine competes with mitochondrial O_SCPLOWLC_SCPLOW-serine transport, depleting the substrate of Shmt2. Functionally, under O_SCPLOWLC_SCPLOW-serine poor conditions in vitro and ex vivo, O_SCPLOWDC_SCPLOW-serine inhibited the proliferation of immature and undifferentiated neural cells including glioblastoma stem cells, which depend highly on one-carbon metabolism. Notably, endogenous O_SCPLOWDC_SCPLOW-serine levels were low during early neurodevelopment, but increased with maturation, coinciding with a shift in the transcriptional profiles of serine metabolic enzymes at the cellular level. Given that O_SCPLOWLC_SCPLOW-serine supports neurodevelopment and O_SCPLOWDC_SCPLOW-serine modulates neurotransmission, this developmental shift in serine enantiomer metabolism appears to align with the functional transitions of the maturing nervous system. Thus, our findings reveal that serine chirality can influence mitochondrial substrate availability and one-carbon flux, offering previously unappreciated insight into the stereoselective regulation of cellular metabolism.

developmental biology↗

bioRxiv2022

Molecular dissection of condensin II-mediated chromosome assembly using in vitro assays

In vertebrates, condensin I and condensin II cooperate to assemble rod-shaped chromosomes during mitosis. Despite recent progress in our understanding of the mechanism of action of condensin I, how condensin II might work has remained largely unexplored. By introducing recombinant condensin II complexes into Xenopus egg extracts, we dissect the roles of its individual subunits in chromosome assembly. We show that two HEAT subunits, CAP-D3 and CAP-G2, play positive and negative roles, respectively, in condensin II-mediated chromosome assembly. The SMC ATPase and the basic amino acid clusters of the kleisin subunit are essential for this process. Deletion of the C-terminal tail of CAP-D3 increases the ability of condensin II to assemble chromosomes and further exposes a hidden function of CAP-G2 in the lateral compaction of chromosomes. Taken together, our results uncover a multilayered regulatory mechanism unique to condensin II, and provide profound implications in the evolution of condensin II.

cell biology↗

bioRxiv2022

Biallelic and gene-wide genomic substitution for endogenous intron and retroelement mutagenesis in human cells

Functional annotation of the vast noncoding landscape of the diploid human genome still remains a major challenge of genomic research. An efficient, scarless, biallelic, and gene-wide mutagenesis approach is needed for direct investigation of the functional significance of endogenous long introns in gene regulation. Here we established a genome substitution platform, the Universal Knock-in System or UKiS, that meets these requirements. For proof of concept, we first used UKiS on the longest intron of TP53 in the pseudo-diploid cell line HCT116. Complete deletion of the intron, its substitution with mouse and zebrafish syntenic introns, and specific removal of retrotransposon-derived elements (retroelements) were all efficiently and accurately achieved in both alleles, revealing a suppressive role of intronic Alu elements in TP53 expression. We also used UKiS for TP53 intron deletion in human induced pluripotent stem cells without losing their stemness. Furthermore, UKiS enabled biallelic removal of all introns from three human gene loci of [~]100 kb and longer to demonstrate that intron requirements for transcriptional activities vary among genes. UKiS is a new standard platform with which to pursue the design of noncoding regions for genome writing in human cells.

bioengineering↗

bioRxiv2018

Characteristics of Interactions at Protein Segments External to Globular Domains in the Protein Data Bank

The principle of three-dimensional protein structure formation is a long-standing conundrum in structural biology. A globular domain of a soluble protein is formed by a network of atomic contacts among amino acid residues, but regions external to globular domains, like loop and linker, often do not have intramolecular contacts with globular domains. Although these regions can play key roles for protein function as interfaces for intermolecular interactions, their nature remains unclear. Here, we termed protein segments external to globular domains as floating segments and sought for them in tens of thousands of entries in the Protein Data Bank. As a result, we found that 0.72 % of residues are in floating segments. Regarding secondary structural elements, coil structures are enriched in floating segments, especially for long segments. Interactions with polypeptides and polynucleotides, but not small compounds, are enriched in floating segments. The amino acid preferences of floating segments are similar to those of surface residues, with exceptions; the small side chain amino acids, Gly and Ala, are preferred, and some charged side chains, Arg and His, are disfavored for floating segments compared to surface residues. Our comprehensive characterization of floating segments may provide insights into understanding protein sequence-structure-function relationships.

bioinformatics↗

bioRxiv2018

Autonomous functionality of an upstream open reading frame in polycistronic mammalian mRNAs

Upstream open reading frames (uORFs) are established as cis-acting elements for eukaryotic translation of annotated ORFs (anORFs) located on the same mRNAs. Here, we identified a mammalian uORF with functions that are independent from anORF translation regulation. Bioinformatics screening using ribosome profiling data of human and mouse brains yielded 308 neurologically vital genes from which anORF and uORFs are polycistronically translated in both species. Among them, Arhgef9 contains a uORF named SPICA, which is highly conserved among vertebrates and stably translated only in specific brain regions of mice. Disruption of SPICA translation by ATG-to-TAG substitutions did not perturb translation or function of its anORF product, collybistin. SPICA-null mice displayed abnormal maternal reproductive performance and enhanced anxiety-like behavior, characteristic of ARHGEF9-associated neurological disorders. This study demonstrates that mammalian uORFs can be independent genetic units, revising the prevailing dogma of the monocistronic gene in mammals, and even eukaryotes.

molecular biology↗